Recombinant antibacterial group iia phospholipase a2 and methods of use thereof

ABSTRACT

Disclose herein is a novel recombinant mutant protein of human Group IIA phospholipase A2 (PLA2) which has enhanced antibacterial activity when compared to the wild-type human Group IIA PLA2, pharmaceutical formulations comprising the protein and methods of use thereof. Additionally, the formulations may comprise other bioactive compounds, such as, e.g., conventional antibiotics, that act additively or synergistically with Group IIA PLA2 in order to promote bacterial killing.

[0001] The United States Government has certain rights to this inventionby virtue of funding received from the U.S. Public Health Service GrantRO1-A1 18571.

FIELD OF THE INVENTION

[0002] This invention pertains to a novel recombinant mutant protein ofhuman Group IIA Phospholipase A2 (PLA2) which has significantly enhancedantibacterial activity compared to the wild-type human Group IIA PLA2,pharmaceutical formulations comprising the protein and methods of usethereof.

BACKGROUND OF THE INVENTION

[0003] The growing prevalence of antibiotic resistance in bacterialpathogens has stimulated renewed interest in the discovery of novelantibiotics. U.S. Pat. No. 5,874,079 discloses that a “Group IIA” 14 kDaPhospholipase A2 (PLA2), mobilized during inflammation expresses potentbactericidal activity toward a broad range of clinically importantGram-positive bacteria and enhances the activity of the host defensemechanisms toward many Gram-negative bacteria.

[0004] The phospholipase A2 (PLA2) family of enzymes hydrolyze the sn-2ester of glycerophospholipids to produce a fatty acid and alysophospholipid (Dennis, J. Biol. Chem. 269:13057-13060, 1994; Gelb etal, Ann. Rev. Biochem. 64, 653-688, 1995; Waite, The phospholipases,Plenum Press, New York, 1987). Based on amino acid sequences, 10 groupsof PLA2s have been identified, including eight from mammals (Dennis,Trends Biochem. Sci. 22: 1-2, 1997; Cupillard et al., J. Biol. Chem.272: 15745-15752, 1997). Group IIA PLA2 in mammals are produced by manydifferent cell types including phagocytic cells, platelets, Paneth cellsand lacrimal cells. It has been shown that both rabbit and human GroupIIA PLA2 can, in concert with other host defense mechanisms, increasethe destruction of gram-negative bacteria (Wright et al., J. Clin.Invest. 85: 1925-1935, 1990; Weiss et al., J. Biol. Chem. 269:26331-26337, 1994 Elsbach et al., Trends Microbiol. 2: 324-328, 1994 andMadsen et al., Infect. Immun. 64: 2425-2430, 1996) and by itself, killmany gram-positive bacteria (Weinrauch et al., J. Clin. Invest. 97:250-257, 1996). The antibacterial activity of Group IIA PLA2 appears tobe a specific attribute of the mammalian 14 kDa isoform. This is furtherexemplified in experimentally induced local inflammatory (ascitic) fluidin rabbits, whereby the mobilization of Group IIA PLA2 is fullyresponsible for the potent bactericidal activity expressed in the fluidtoward S. aureus and several other gram-positive bacteria (Weiss et al.,en supra). Normal plasma, by contrast contains low levels of PLA2 andantistaphylococcal activity. It has recently been shown that themobilization of this enzyme in baboons during inflammation may play animportant role in host defense mechanisms against invading bacteria(Weinrauch et al., J. Clin. Invest. 102 (3): 633-638, 1998).

[0005] In biological fluids, as little as 100 ng/ml of the human GroupII A PLA2 is sufficient to kill greater than 99% of 10⁶ Staphylococcusaureus cells/ml, including all multi-drug resistant clinical isolatestested. The bactericidal activity of the PLA2 was dependent on catalyticactivity and was enhanced synergistically by the co-treatment withsub-inhibitory doses of β-lactam antibiotics. The potent antibacterialactivity of the mammalian Group IIA PLA2 is not expressed by otherclosely related PLA2s reflecting the presence and localization of a highdensity of basic residues in the Group IIA PLA2 that is absent in allother subsets of related PLA2s.

[0006] U.S. Pat. No. 5,874,079 discloses that the rabbit Group IIA PLA2possesses 10 fold greater antibacterial activity than the human enzyme.Since it is preferable to treat humans with human-derived therapeuticproteins, what is needed is a human PLA2 with activity similar to therabbit counterpart.

SUMMARY OF THE INVENTION

[0007] In one aspect, the present invention provides methods fortreating Gram-positive bacterial infections in humans, by administeringbactericidal-effective amounts of mutant human Group IIA PLA2.

[0008] In another aspect, the invention provides pharmaceuticalformulations having bactericidal activity against Gram-positivebacteria. These formulations comprise bactericidal-effectiveconcentrations of mutant human Group IIA PLA2 and a pharmaceuticallyacceptable carrier or diluent. Additionally, the formulations maycomprise other bio active compounds, such as, e.g., conventionalantibiotics, that act additively or synergistically with Group IIA PLA2to promote bacterial killing.

[0009] These and other aspects of the present invention will be apparentto those of ordinary skill in the art in light of the presentdescription, claims and drawings.

BRIEF DESCRIPTION OF DRAWINGS

[0010] FIGS. 1(A, B, C and a,b,c) are space-filling models of wild-typehuman (FIGS. 1A, a), rabbit (FIGS. 1B, b) and mutant (G72K.T103K, FIG.1C, c) human Group IIA PLA2 showing the distribution of charged residueswithin these PLA2. Models are based on the solved X-ray structure ofhuman Group IIA PLA2 (Scott, D. L., White, S. P., Browning, J. L., Rosa,J. J., Gelb, M. H., Sigler, P. B. 1991. Science 254: 1007-1010). Sitesshown in black represent basic residues (arg and lys), sites in graycorrespond to acidic residues (asp and glu). All other residues aredisplayed in white. Two images of each enzyme are shown corresponding toapproximately 180 degree rotations (a, b,c), for WT human, WT rabbit andmutant human, respectively. Note that residues are numbered in acontinuous fashion according to the primary structures of these PLA2s.

[0011]FIG. 2 is a graphic illustration of the comparison of bactericidalactivity of native human and rabbit Group II A PLA2 towards S. aureus52A.

[0012] FIGS. 3(A and B) show the DNA (A), SEQ. ID. NO. 1 and the protein(B), SEQ. ID. NO. 2 sequence, respectively, of mutant human Group IIAPLA2. Residues differing from sequence of wild-type human Group IIA PLA2are shown in bold and underlined.

[0013]FIG. 4 is a graphic illustration of the comparison of thebactericidal activity of human wild-type (WT) and mutant human and WTrabbit Group II A PLA2 toward S. aureus RN450.

[0014]FIG. 5 is a graphic illustration of the comparison of thebactericidal activity of human wild-type (WT), and mutant human and WTrabbit Group II A PLA2 towards S. aureus RN450 and two clinicalisolates, strains 5A and 18 S. aureus.

[0015] FIGS. 6(A, B and C) are graphic illustrations showing WT humanand WT rabbit and mutant human Group II A PLA2-induced killing of S.aureus strains expressing 1(A) 5(B) or 8(C) capsular serotypepolysaccharides.

[0016] FIGS. 7(A and B) is a graphic illustration showing thatencapsulated (A) and non-encapsulated (B) S. aureus were equallysusceptible to killing by a whole inflammatory fluid and that killing ofboth strains was prevented by neutralizing antiserum to Group IIA PLA 2.

[0017]FIG. 8 is a graphic illustration showing that the killing ofencapsulated S. aureus by whole inflammatory exudates is blocked byGroup IIA PLA2 antiserum.

[0018]FIG. 9 is a graphical illustration showing that the mutant humanGroup IIA PLA 2 enzyme accelerates clearance of S. aureus infection invivo.

[0019]FIG. 10 is a graphical illustration showing that the mutant humanGroup II A PLA 2 reduces S. aureus bacteremia.

[0020]FIG. 11 is a graphical illustration showing that administration ofthe mutant human Group IIA PLA 2 reduces metastatic kidney infectioncaused by S. aureus.

DETAILED DESCRIPTION OF THE INVENTION

[0021] All patent applications, patents, and literature references citedin this specification are hereby incorporated by reference in theirentirety.

[0022] Definitions:

[0023] 1. “Nucleic acid” or “polynucleotide” as used herein refers topurine- and pyrimidine-containing polymers of any length, eitherpolyribonucleotides or polydeoxyribonucleotides or mixedpolyribo-polydeoxyribo nucleotides. This includes single- anddouble-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids,as well as “protein nucleic acids” (PNA) formed by conjugating bases toan amino acid backbone. This also includes nucleic acids containingmodified bases.

[0024] 2. An “isolated” nucleic acid or polypeptide as used hereinrefers to a nucleic acid or polypeptide that is removed from itsoriginal environment (for example, its natural environment if it isnaturally occurring). An isolated nucleic acid or polypeptide containsless than about 50%, preferably less than about 75%, and most preferablyless than about 90%, of the cellular components with which it wasoriginally associated.

[0025] 3. A nucleic acid or polypeptide sequence that is “derived from”a designated sequence refers to a sequence that corresponds to a regionof the designated sequence. For nucleic acid sequences, this encompassessequences that are homologous or complementary to the sequence, as wellas “sequence-conservative variants” and “function-conservativevariants.” For polypeptide sequences, this encompasses“function-conservative variants.” Sequence-conservative variants arethose in which a change of one or more nucleotides in a given codonposition results in no alteration in the amino acid encoded at thatposition. Function-conservative variants are those in which a givenamino acid residue in a polypeptide has been changed without alteringthe overall conformation and function of the native polypeptide,including, but not limited to, replacement of an amino acid with onehaving similar physico-chemical properties (such as, for example,acidic, basic, hydrophobic, and the like). “Function-conservative”variants also include any polypeptides that have the ability to elicitantibodies specific to a designated polypeptide.

[0026] In general, nucleic acid manipulations used in practicing thepresent invention employ methods that are well known in the art, asdisclosed in, e.g., Molecular Cloning, A Laboratory Manual (2nd Ed.,Sambrook, Fritsch and Maniatis, Cold Spring Harbor) and CurrentProtocols in Molecular Biology (Eds. Ausubel, Brent, Kingston, More,Feidman, Smith and Stuhl, Greene Publ. Assoc., Wiley-Interscience, NY,N.Y., 1997).

[0027] The present invention is directed to methods and compositions forkilling Gram-positive bacteria that take advantage of the bactericidalaction of Group IIA phospholipase A2 (PLA2). In practicing theinvention, bacteria are exposed to or contacted with, a Gram-positivebactericidal-effective amount of mutant human Group IIA PLA2, resultingin the rapid inactivation and death of Gram-positive bacteria. Accordingto the invention, bacterial infections in humans can be treated byadministering mutant human Group IIA PLA2. The invention alsoencompasses pharmaceutical formulations suitable for therapeuticadministration.

[0028] The mutant human Group II A PLA2 of the present invention has 10×greater antibacterial activity and a net charge +2 greater than thehuman WT homologue (+17 vs. +15). Based on the comparison of thestructural and functional properties of rabbit and human wild-type andmutant Group II A PLA2 (see FIG. 1), discrete alterations of human Group11 A PLA2 by site-specific mutagenesis yielded novel human Group II APLA2 proteins (herein referred to as “mutant”) with markedly enhancedantibacterial activity which may be measured using any procedurewell-known in the art, including that described in Example 3 below.

[0029] Previous studies with site-specific mutants of human Group IIAPLA2 indicated that the high net (+) charge of Group IIA PLA2 inaddition to its catalytic properties were essential for the potentantibacterial properties of Group IIA PLA2. The high net (+) charge andantibacterial properties are unique attributes of this subset of PLA2.In addition, comparison of the structural and functional properties ofnative and recombinant rabbit and human Group IIA PLA2 demonstratedhigher antibacterial activity in the rabbit PLA2 along with a higher net(+) charge (+17 for the rabbit enzyme, +15 for the human PLA2).Following cloning of the cDNA encoding rabbit Group IIA PLA2 andsequence analysis, comparison of the primary structures of rabbit andhuman Group IIA PLA2 revealed 32 sites that were different in these twoenzymes (26% of 124 residues; no gaps). Of these, at least 22represented relatively conservative substitutions. Two differences stoodout; arginine (rabbit) vs. glycine (human) at residue 72 and lysine(rabbit) vs. threonine (human) at residue 103. These differences accountfor the higher net charge of the rabbit PLA2 and were located along ahighly cationic ridge on the enzyme surface (see FIG. 1). Mutagenesisstudies indicated that the charge properties of this region areessential for potent antibacterial activity and also suggested that thecharge properties of other regions may not be equally important forantibacterial activity. Consequently, residues 72 and 103 were chosenfor mutagenesis. It is possible that introduction of basic residueseither at other surface sites within this highly cationic ridge oroutside this region, even at sites that do not normally contain basicamino acids in the native Group IIA PLA2, could also confer increasedantibacterial activity.

[0030] It should be noted that among all the “low Mr” (i.e. 13-18 kDa)PLA2, including the native Group IIA PLA2, the density and distributionof charged residues along the enzyme surface varies widely withoutaffecting overall protein conformation and catalytic activity towardartificial substrates. This is also true in surface charge changesintroduced genetically in PLA2 variants. Thus, great variation in theenzyme surface charge can be well tolerated, affording ample opportunityto create many permutations of enzyme structure that may enhanceantibacterial potency.

[0031] In a preferred embodiment, E. coli is stabily transformed with acDNA encoding mutant human Group IIA PLA2, as set forth in FIG. 3A andis used as a recombinant source of Group IIA PLA2 (see Example 1). Forrecombinant expression, Group IIA PLA2-encoding DNA, contained within aDNA vector, must be operably linked to a transcriptional promoter sothat functional Group IIA PLA2 mRNA is transcribed and Group IIA PLA2protein is synthesized within the transformed host cell. Preferably, therecombinant protein is recovered in E. coli inclusion bodies and thenthe expressed protein is subjected to in vitro reversibledenaturation/reduction followed by renaturation/oxidation to promoteproper disulfide bond formation.

[0032] The invention also encompasses vectors comprising mutant humanPLA2-encoding sequences, cells comprising the vectors, and methods forproducing mutant human PLA2 that involve culturing the cells.

[0033] A large number of vectors, including plasmid and fungal vectors,have been described for expression in a variety of eukaryotic andprokaryotic hosts. Advantageously, vectors may also include a promotoroperably linked to the mutant human PLA2 encoding portion. The encodedmutant human PLA2 may be expressed by using any suitable vectors andhost cells, using methods disclosed or cited herein or otherwise knownto those skilled in the relevant art. The particular choice ofvector/host is not critical to the invention.

[0034] Vectors will often include one or more replication systems forcloning or expression, one or more markers for selection in the host,e.g. antibiotic resistance, and one or more expression cassettes.Ligation of the coding sequences to the transcriptional regulatorysequences may be achieved by known methods. Suitable host cells may betransformed/transfected/infected by any suitable method includingelectroporation, CaCl₂ mediated DNA uptake, fungal infection,microinjection, microprojectile, or other established methods.

[0035] Appropriate host cells include bacteria, archebacteria, fungi,especially yeast, and plant and animal cells, especially mammaliancells. Of particular interest are E. coli, B. subtilis, S. cerevisiae,SF9 cells, C129 cells, 293 cells, Neurospora, CHO cells, COS cells, HeLacells, and immortalized mammalian myeloid and lymphoid cell lines.Preferred replication systems include M13, ColE1, SV40, baculovirus,lambda, adenovirus, and the like. A large number of transcriptioninitiation and termination regulatory regions have been isolated andshown to be effective in the transcription and translation ofheterologous proteins in the various hosts. Examples of these regions,methods of isolation, manner of manipulation, etc. are known in the art.Under the appropriate expression conditions, host cells can be used as asource of recombinantly produced mutant human PLA2.

[0036] Nucleic acids encoding mutant human PLA2 polypeptides may also beintroduced into cells by recombination events. For example, such asequence can be introduced into a cell, and thereby effect homologousrecombination at the site of an endogenous gene or a sequence withsubstantial identity to the gene. Other recombination-based methods,such as non-homologous recombinations or deletion of endogenous genes byhomologous recombination, may also be used.

[0037] The invention also encompasses isolated and purified mutant humanPLA2 polypeptides, including, e.g., a polypeptide having the amino acidsequence depicted in FIG. 3B, as well as function-conservative variantsof this polypeptide, including fragments that retain antibacterialactivity as described above.

[0038] Purification of mutant human Group IIA PLA2 from recombinantsources may be achieved by methods well-known in the art, includingwithout limitation ion-exchange chromatography, reversed-phase highperformance liquid chromatography (HPLC) on C4 columns, gel filtration,isoelectric focusing, affinity chromatography, immunoaffinitychromatography, and the like. For some purposes, it is preferable toproduce the polypeptide in a recombinant system in which the proteincontains an additional sequence tag that facilitates purification, suchas, but not limited to, a polyhistidine sequence. The polypeptide canthen be purified from a crude lysate of the host cell by chromatographyon an appropriate solid-phase matrix.

[0039] In a preferred embodiment, a cell-free fluid containing mutanthuman Group IIA PLA2 is subjected to ion-exchange chromatography onSP-Sepharose, followed by reversed-phase high performance liquidchromatography (HP-LC) on a C4 column. Purity of the final Group IIAPLA2 preparations is confirmed by SDS-PAGE and by OD at 280 m, using theknown extinction coefficient for this protein (OD of 1.0=0.9 mg/ml).

[0040] The isolated polypeptide may be modified by, for example,phosphorylation, sulfation, acylation, or other protein modifications.It may also be modified with a label capable of providing a detectablesignal, either directly or indirectly, including, but not limited to,radioisotopes and fluorescent compounds.

[0041] According to the present invention, the mutant human Group IIAPLA2 of the present invention is characterized by bactericidal activityagainst gram positive bacteria, such as S. aureus (see Example 3).Antibacterial activity of mutant human Group IIA PLA2 may be quantifiedby measuring the colony-forming ability cf susceptible bacteria thathave been incubated with or without increasing amounts of mutant humanGroup IIA PLA2. Typically, a suspension of 10⁶ bacteria/ml (e.g., S.aureus) is exposed to 1-500 ng/ml of mutant human Group IIA PLA2 for60-90 minutes at 37° C., after which the cells are mixed with moltenagar and plated. After overnight growth, bacterial colonies are comparedbetween mutant Group IIA PLA2-treated and untreated cultures.

[0042] The present invention encompasses, in addition to the mutanthuman Group IIA PLA2 disclosed herein, other recombinant forms of GroupIIA PLA2 that are formed by site-specific genetic manipulations and havedetectable Gram-positive bactericidal activity. The methods andcompositions of the present invention encompass any deletion, addition,or substitution mutant of Group IIA PLA2 produced by the methodsdescribed herein that increase the wild-type enzymatic and antibacterialactivity.

[0043] It will be understood that the methods for expression,purification, and activity measurements described above for the mutanthuman Group IIA PLA2 can also be applied to variant Group IIA PLA2species. Thus also, only routine experimentation is required to identifyadditional, new useful Group IIA PLA2 variants.

[0044] Therapeutic Applications

[0045] The enhanced antibacterial potency of the mutant human Group II APLA2 protein described herein provides new therapeutic approaches to thetreatment of potentially life-threatening infections caused bymulti-drug resistant Gram-positive bacteria. The applications includewound and bloodstream infections with methicillin-resistant S. aureus(MRSA) and nosocomial infections with vancomycin-resistant Enterococcusfaecium. These infections are much more common and potentially lifethreatening in immunocompromised or hospitalized patients. The abilityof the Group II A PLA2 to act in synergy with various β-lactamantibiotics and, most importantly, with otherwise β-lactam-resistantbacteria (e.g. MRSA), also allows for the use of the PLA2 of the presentinvention in conjunction with antibiotics otherwise rendered ineffectiveby the growing prevalence of antibiotic resistance.

[0046] According to the present invention, recombinant mutant humanGroup IIA PLA2 may be formulated with a physiologically acceptablecarrier, such as, for example, phosphate buffered saline or deionizedwater. The pharmaceutical formulation may also contain excipients,including preservatives and stabilizers, that are well-known in the art.The compounds can be formed into dosage units such as, for example,liquids, tablets, capsules, powders, suppositories, and may additionallyinclude excipients that act as lubricant(s), plasticizer(s),colorant(s), absorption enhancer(s), bactericide(s), and the like. Thedosage forms may contain mutant human Group IIA PLA2 at concentrationsranging between about 100 ng/ml and about 100 μg/ml. Solid dosage formssuch as tablets and powders may contain the mutant human Group IIA PLA2of the present invention at appropriate concentrations so thatbactericidal effective amounts of mutant human Group IIA PLA2 (seebelow) can be delivered using conventional administration regimens. Itwill be understood that the pharmaceutical formulations of the presentinvention need not in themselves contain the entire amount of the agentthat is effective in treating the disorder, as such effective amountscan be reached by administration of a plurality of doses of suchpharmaceutical formulations.

[0047] Modes of administration of the mutant human Group IIA PLA2 of thepresent invention to achieve a therapeutic benefit include topical, oraland enteral, intravenous, intramuscular, subcutaneous, transdermal,transmucosal (including rectal and buccal), and by inhalation routes.Generally, Group IIA PLA2 and specifically the mutant human Group IIAPLA2 of the present invention are extremely stable proteins and toleratea wide variety of environmental conditions. It will be understood thatthe mode of administration will depend on the nature of the syndrome,including the location and severity of the Gram-positive bacterialinfection. For example, skin lesions may be treated using a topicalointment, whereas a bacteremia may require intravenous administration.An internal but localized infection may be treated by injecting theformulation directly into the site of the infection.

[0048] An “effective amount” of the mutant human Group IIA PLA2 of thepresent invention for treating a particular bacterial infection is anamount that results in a detectable reduction in the severity of theinfection. This may be measured directly, i.e., by counting or culturingthe pathogenic microorganisms, or indirectly, by monitoring clinicalsigns of infection, such as fever or purulent discharge. Typically,administration of the mutant human Group IIA PLA2 will result in thelessening or amelioration of at least one symptom of the infection. Anyamelioration resulting from administration of the mutant human Group IIAPLA2 of the present invention of any symptom of infection is within thescope of the invention. The effective amount for treating a givensyndrome in a human can be determined by routine experimentationwell-known in the art, such as by establishing a matrix of dosages andfrequencies and comparing a group of experimental units or subjects toeach point in the matrix.

[0049] An additional consideration in establishing the optimum dosage ofthe mutant human Group IIA PLA2 for treating bacterial infections ispotential toxicity. Though there have been reports that Group IIA PLA2possesses inflammatory activity (see, for example, Bomalaski et al., J.Immunol. 146:3904, 1991; and Cirino et al., J. Rheumatol. 21:824, 1994),this phenomenon was only observed at Group IIA PLA2 concentrationsseveral orders of magnitude higher than those at which bactericidaleffects are observed. Furthermore, the preparations used in the studiescited above were contaminated with endotoxin, which itself has a potentinflammatory activity. Thus, without wishing to be bound by theory, itis believed that bactericidal effective amounts of Group IIA PLA2 can beadministered to humans without causing inflammatory or other detrimentalside effects. In addition, it is preferable to treat humans with GroupIIA PLA2 derived from the same species.

[0050] The effective amount of the mutant human Group IIA PLA2 of thepresent invention for treating infections caused by gram-positivebacteria to be administered may range between about 1 and about 100μg/kg/body weight, preferably from about 1 to about 10 μg/kg/bodyweight. In a preferred embodiment, mutant human Group IIA PLA2 of thepresent invention is formulated in a sterile saline solution, which isadministered intravenously to a patient suffering from anantibiotic-resistant S. aureus bacteremia.

[0051] In another embodiment, an antibacterial formulation is preparedcontaining, in addition to the mutant human Group IIA PLA2, otherconventional antibiotics (such as, for example, β-lactam antibiotics),or other bioactive substances, that may act additively orsynergistically with the mutant human Group IIA PLA2 of the presentinvention to kill Gram-positive bacteria. It is believed that the use offormulations containing the mutant human Group IIA PLA2 of the presentinvention in conjunction with, for example, sub-lethal doses of otherantibiotics would provide a clinical advantage in reducing the overalladministration of antibiotics (thus lessening the development ofantibiotic-resistant strains).

[0052] Antibiotics that can be used in conjunction with mutant humanGroup IIA PLA2 of the present invention in the methods and compositionsof the present invention include without limitation penicillins (such asampicillin, amoxicillin, oxacillin, and the like), cephalosporins,aminoglycosides (such as streptomycin, neomycin, kancmycin, gentamicin,and the like), tetracyclines, chloramphenical, and vancomycin.Commercial sources for each of these are presented in Table I below.TABLE 1 Drug Source City, State Ampicillin Warner Chilcott LaboratoriesRockaway, NJ Amoxicillin Warner Chilcott Laboratories Rockaway, NJOxacillin Teva Pharmaceuticals Sellersville, PA Cefotaxime HoechstMarion Roussel Kansas City, MO Streptomycin Pfizer New York, NY NeomycinMerck West Point, PA Kanamycin SolePak Pharmaceuticals Boca Raton, FLGentamicin SoloPak Pharmaceuticals Boca Raton, FL Tetracycline LederleStandard Products Philadelphia, PA Chloramphenicol Fujisawa Deerfield,IL Vancomycin Eli Lilly and Co. Indianapolis, IN

[0053] As shown below in Example 7, the human mutant Group IIA PLA2 ofthe present invention was effective in treating S. aureus infection invivo. Given the rapidly spreading occurrence of drug resistant bacteria,the mutant enzyme of the present invention will provide a usefuladdition to the arsenal of presently used anti-(Gram positiveantibiotics.

[0054] The following examples are intended to further illustrate theinvention without limiting the scope thereof.

EXAMPLE 1 Preparation of Mutant (G72KT103K) Human Group II A PLA2 cDNA

[0055] cDNA encoding the mutant human Group II A phospholipase A2 wasproduced by mismatched primer PCR using oligonucleotide primers directedagainst wild-type human Group II PLA 2 cDNA (clone hp PLA2 9-1)subcloned within pEE14 (CellTech) as described in Weiss, et al., 1994,J. Biol. Chem. 269: 26331-26337. The oligonucleotide primer 5′TTTGCTAGAAACAAGAAGACCTACAAT 3′ has a single base pair change (underlinedand in bold) conferring the threonine to lysine substitution at residue103 and is complementary to codons 98-106 of the non-coding strand ofthe human Group II A phospholipase A2. The single mutant T103K wascreated by substitution of lysine for threonine at residue 103. Thedouble mutant G72K.T103K was constructed on the DNA template of theT103K mutant using the primer 5′ TTTAGCAACTCGAAGAGCAGAATCACC 3′ which iscomplementary to the non-coding strand from residues 68-76 with theindicated two base change to produce the glycine to lysine substitutionat residue 72. Furthermore, to facilitate purification of recombinantGroup II A PLA2, expressed as part of a fusion protein, one additionalsubstitution was introduced (L8M) using the oligonucleotide primer 5′GCTCGAGATGAATTTGGTGAATTTCCACAGACTGATC 3′ which is complementary to thenon-coding strand of the human Group II A PLA2 from codons 1-9. Thisalteration eliminates the single methionine amino acid residue withinthe PLA2 coding region permitting excision of intact PLA2 from thefusion partner by CNBr treatment.

EXAMPLE 2 Expression and Purification of Mutant (G72K.T103K) Human GroupIIA PLA2

[0056] The mutated Group II A PLA2 cDNA was subcloned into Xho I andEcoRI restriction sites of E. coli expression vector pRSETA (Invitrogen)and expressed as a fusion protein under the control of the bacteriophageT7 promoter. Expression of the recombinant protein was induced by thetreatment of transformed E. coli BL21 (DE3) with IPTG. The recombinantprotein was recovered in inclusion bodies, extracted and modified byS-sulfonation as previously described. (Thannhauser et al., Biochemistry24 7681-7688., 1985; Liang, N. S. et al. FEBS Letters 334:55-59. 1993;Fourcade et al., Cell 80: 919-923, 1995). The S-sulphonated protein wasprecipitated by dialysis against 1% acetic acid and cleaved by cyanogenbromide to release mature Group IIA PLA2 from the fusion protein.Refolding and disulfide bond formation of recombinant Group IIA PLA2 wascarried out in the cold for 72 hours in 10mM sodium borate buffer (pH8.5) containing 2 mM cystine, 10 mM cysteine, 10 mM CaCl₂ and 0.85 Mguanidinium hydrochloride followed by dialysis against 50 mM sodiumacetate/acetic acid buffer, pH 5.0. Refolded, active Group IIA PLA2 waspurified from improperly folded, inactive protein by chromatography onSP-Sepharose and reversed-phase high performance liquid chromatography(HPLC) on a C4 column. The purity of the recovered Group IIA PLA2 wasconfirmed by analytical reversed-phase HPLC and by absorbence at 280 nm,using the known extinction coefficient for this protein (OD of 1.0=0.9mg/ml).

EXAMPLE 3 Comparison of the Bactericidal Activity of Wild-Type andRecombinant Human, and Rabbit Group IIA PLA2s against Staphylococcusaureus 52A

[0057] The bactericidal activity of wild-type human and recombinantmutant human and rabbit Group IIA PLA2s against Staphylococcus aureus52A (106 per ml)was determined. S. aureus was incubated with 0.01-100 nMof each Group IIA PLA2 and incubated at 37° C. for 2 hours in RPMI-1640medium containing 10 mM HEPES, pH 7.4 and 1% (w/v) albumin. Afterincubation, bacterial viability was determined by measuring the colonyforming ability of the bacteria in trypticase soy agar. Bacterialcolonies were enumerated after 18-24 hours at 37° C. The results areexpressed as the percentage of the colony forming units (CFU) of theoriginal bacterial inoculum (i.e. at T=0).

[0058] The graphical results depicted in FIG. 2 show that about 10-foldlower concentrations of rabbit PLA2 than of human PLA2 suffice toproduce the same reduction of CFU. Therefore, rabbit Group IIA PLA2 hada greater bactericidal effect on S. aureus cells than the human enzyme.

EXAMPLE 4 Comparison of the Bactericidal Activity of Wild-Type (WT) andRecombinant Human, and Rabbit Group II A PLA2 toward S. aureus RN450.

[0059] The bactericidal activity of wild-type and recombinant mutanthuman and rabbit Group IIA PLA2s against Staphylococcus aureus RN450 wasdetermined.

[0060]S. aureus RN450 (10⁶/ml) was incubated with 1-500 ng/ml of eachGroup IIA PLA2 at 37° C. for 60 minutes in RPMI-1640 medium containing10 mM HEPES, pH 7.4 and 1% (w/v) albumin. Bacterial viability wasdetermined as in Example 3.

[0061] The graphical results depicted in FIG. 4 show that thebactericidal activities of recombinant mutant human Group IIA PLA2 andWT rabbit Group IIA PLA2 are nearly equivalent. Furthermore, thegenetically modified mutant human Group IIA PLA2 had increasedantibacterial activity against S. aureus compared to wild-type humanGroup IIA PLA2.

EXAMPLE 5 Comparison of the Bactericidal Activity of Recombinant Humanand Rabbit Group II A PLA2 toward S. aureus RN450 and Two ClinicalIsolates, Strains 5A and 18.

[0062]S. aureus RN450 and two clinical isolates, strains 5A (MRSA) and18 (10⁷/ml were incubated with 1-500 ng/ml of each Group IIA PLA2 at 37°C. for 90 minutes in 70% (v/v) pooled human sera diluted in Hanks'balanced salts solution and buffered with 10 mM HEPES, pH 7.4.

[0063] As shown in FIGS. 4 and 5, the mutant human Group IIA PLA2 wasessentially as active as the wild-type rabbit enzyme toward each of theseveral strains of S. aureus, and, hence, significantly more active thanthe wild-type human Group IIA PLA2. This is manifested in both anartificial laboratory medium (FIG. 4) and in an environment that moreclosely simulates that of circulating body fluids (FIG. 5).

[0064] These findings further demonstrate that the novel mutant GroupIIA phospolipase A2 described herein represents a more potentantibacterial product than the wild-type human enzyme.

EXAMPLE 6 Encapsulated and Non-Encapsulated S. aureus are Killed byGroup IIA PLA2

[0065] One setting in which mobilization of extracellular Group IIA PLA2may be particularly important is against encapsulated strains of S.aureus. The majority of bacteremic isolates of S. aureus areencapsulated (Hochkeppel et. al., J. Clin. Microbiol. 25:526-530) andbacteria recovered from more chronically infected sites as in cysticfibrosis are also covered with an extracellular carbohydrate polymer(McKenney et al., Science 284:1523-1527). In the absence oftype-specific anti-capsular antibodies, encapsulated strains arerelatively resistant to opsonophagocytic killing by PMN (Xu et al.,Infect. Immun. 60:1358-1362; Table I). In contrast, purified Group IIAPLA2 and PLA2 present in elicited inflammatory fluids exhibit equipotentbactericidal activity against encapsulated S. aureus and isogenicnon-encapsulated derivates (FIGS. 6, 7). Moreover, despite inability ofPMN within experimentally elicited acute inflammatory exudates toefficiently ingest encapsulated S. aureus (Table 2), the bacteria arestill top efficiently killed by the inflammatory exudates in anextracellular and PLA2-dependent fashion (FIG. 8). Thus, Group IIA PLA2can provide a potert extracellular weapon against phagocytosis-resistantencapsulated bacteria that is fully active in inflammatory exudates.TABLE 2 EFFECT OF CAPSULE ON SUSCEPTIBILITY OF S. AUREUS TO PHAGOCYTOSISBY RABBIT PMN Strain Bacteria/100 PMN 1B (non-encapsulated) 43.5 1C(encapsulated) 2.3

[0066]S. aureus type IC (encapsulated) and an isogeenic non-encapsulatedderivative (type 1B) were incubated for 30 min. with rabbit pentanealexudate PMN at bacteria/PMN ratio of 2:5 (10⁶ bacteria and 2.5×10⁶PMN/ml). At end of incubation, suspensions were diluted, smears preparedby cytospin and stained. PMN-associated bacteria were visualized bylight microscopy and counted. Results are expressed as number ofbacteria associated with PMN/100 PMN counted. Results indicate a 100%uptake of non-encapsulated bacteria and <10% uptake of encapsulatedbacteria. Similar results were obtained when incubations were carriedout in HEPES-bufferred Hanks' balanced salt solution or in PLA₂-depletedascitic fluid.

EXAMPLE 7 Animal Experiment to Test Efficacy of Administered HumanMutant Group IIA PLA2 against S. aureus Infection in vivo.

[0067] Materials and Methods

[0068] In the example presented below, the following materials andmethods were used.

[0069] Animals: CD/1mice

[0070] Bacteria: Reynolds strain of Staphylococcus aureus (encapsulated;grown overnight on Columbia agar to maximize encapsulation as inexperiments shown in Example 6).

[0071] Administration of bacteria: 2×10⁷/ml in 0.2 ml of sterile RPMIsupplemented with 10 mM HEPES (pH 7.4) and 1% bovine serum albumin.Intraperitoneal (i.p.) inoculation.

[0072] Adminstration of PLA2: 15 μg/0.2 ml of above medium i.p. approx.10-15 min after inoculation of bacteria. Control animals received mediumalone. Enzyme administered was Mutant [G72K.T103K] Group IIA PLA 2.

[0073] Assays of course of infection:

[0074] a) @30, 60, 120, and 240 min after infection, blood samples takenfrom tail vein from each of 4 animals in control and PLA2-treatedgroups. There were a total of 16 animals in each group; each animal wasbled only once in this time period. Levels of bacteremia were assessedby measurement of bacterial CFU in blood.

[0075] b) @1 day after infection, 8 animals from each group weresacrificed. Blood was collected again to measure bacteremia and theperitoneal cavity was washed and inspected to look macroscopically forabscesses (none seen) and measure intraperitoneal bacteria by assay ofCFU.

[0076] c) @ 6 days after infection, the remaining 8 animals from eachgroup were sacrificed and infection in blood and peritoneal cavity wasmeasured as above (still no abscesses seen). This animal model produceslocal and disseminated infection in control animals that is eventuallyself limiting. The level of metastatic infection is greatest at approx.6 days. In addition, kidneys were excised, weighed (no significantdifference between animals within and between treatment groups) andhomogenized to facilitate assay of bacterial CFU within infected kidney(i.e. representing metastatic infection). Abscesses were seen in two of16 control animals; none were seen in PLA2-treated animals.

[0077] The results are shown in FIGS. 9, 10 and 11 for b, a and c,respectively, above.

What is claimed is:
 1. A method for killing Gram-positive bacteria in ahuman patient which comprises contacting said bacteria with abactericidal-effective amount of mutant human Group IIA PLA2.
 2. Themethod of claim 1 wherein said mutant human Group IIA PLA2 isgenetically altered to produce a recombinant protein with amino acidsubstitutions of lysine for glycine at residue 72 and lysine forthreonine at residue
 103. 3. The method of claim 1, wherein saidbacteria are selected from the group consisting of Micrococcus,Staphylococcus, Streptococcus, Peptococcus, Peptostreptococcus,Enterococcus, Methanobacterium, Bacillus, Clostridium, Lactobacillus,Listeria, Erysipelothrix, Corynebacterium, Propionibacterium,Eubacterium, Actinomyces, Arachnia, Bifidobacterium, Bacterionema,Rothia, Mycobacterium, Nocardia, Streptomyces, and Micropolyspora. 4.The method of claim 1, wherein said bacteria are contacted with betweenabout 1 and about 100 μg per kg body weight of said human patient ofmutant human Group IIA PLA2.
 5. A method for killing Staphylococcusaureus bacteria which comprises contacting said bacteria with abactericidal-effective amount of mutant Group IIA PLA2.
 6. A method fortreating a Staphylococcus aureus infection in a human which comprisesadministering to said human an amount effective for treating saidinfection of mutant human Group IIA PLA2.
 7. The method of claim 6wherein said amount effective for treating said infection ranges betweenabout 1 and about 100 μg/kg body weight of said human.
 8. Apharmaceutical formulation comprising mutant human Group IIA PLA2 and apharmaceutically acceptable carrier or diluent, said formulation havingbactericidal activity against Gram-positive bacteria.
 9. The formulationof claim 8 further comprising a β-lactam antibiotic.
 10. A purified,isolated nucleic acid comprising the sequence as set forth in SEQ. IDNo.
 1. 11. A purified, isolated protein comprising the amino acidsequence as set forth in SEQ. ID No.
 2. 12. A method for treating ahuman patient suffering from an infection caused by a Gram-positivebacteria comprising administering to a human in need of such treatment:(a) mutant human Group II PLA2; and (b) an antibiotic, the amounts of(a) and (b) together being effective to treat said infection.
 13. Themethod of claim 12, wherein said antibiotic is selected from the groupconsisting of ampicillin, amoxicillin, oxacillin, cephalosporins,streptomycin, neomycin, kancmycin, gentamicin, tetracyclines,chloramphenical, and vancomycin.